We have found previously that 5-Azacytidine (5-AZA) induces and Transforming Growth Factor (TGF)-b inhibits differentiation of human rhabdomyosarcoma (RMS) cells in vitro. The effect of 5-AZA was associated with increased levels of expression of the muscle determination gene Myo(D1). However, remarkable changes in the expression of the Myo(D1) gene after addition of 1 ng/ml (4 x 10(-11) M) TGF-b were not observed. Although in a recent study Myo(D1) expression has been found repressed by TGF-b1, this was achieved at a 100-fold higher concentration of the agent, and was demonstrated in myoblasts derived from a 5-AZA treated pluripotential cell line and not RMS. In other studies, it was found that TGF-b at a concentration of 4xlO(-10) M blocks the appearance of functional slow Ca2+ channels in differentiated myocytes by inhibiting the expression of the dihydropyridine receptor (DHPR) gene, which is skeletal muscle specific. The expression of Myo(D1) gene was not influenced. We have obtained the DHPR cDNA and we will investigate changes of the expression of this gene, and Myo(D1) in 5 human rhabdomyosarcoma cell lines treated with various concentrations of TGF-b. Although it is not known if the DHP-sensitive Ca2+ channels are necessary events in the myogenic differentiation, extracellular Ca2+ is necessary for muscle development. Possible detectable changes of the DHPR gene in relation to the action of TGF-B and the expression of the Myo(D1) gene will suggest that Ca2+ channels may be part of the intracellular mechanisms that control myogenesis.